This proposal aims to characterize the regulatory hierarchy mediating 20-hydroxyecdysone (20E) action in the adult mosquito. During the first five years of this project we achieved a fundamental understanding of the molecular basis of 20E action in mosquito vitellogenesis. Now, we can ask how this ecdysteroid- regulated hierarchy is adapted for control by a blood meal. We demonstrated the DNA binding properties of the mosquito ecdysteroid receptor complex with respect to its sequence recognition and identified natural response elements in the Aedes and Anopheles genes. We found unique properties of mosquito Ecdysteroid Receptor (EcR) with respect to recognition of ecdysone and 20E. We plan to elucidate determinants in the EcR protein responsible for recognition of ligands and heterodimerization. We will clone two transcription units of the ultraspiracle (usp) gene, encoding USP-A and USP-B isoforms, and elucidate how 20E exerts an opposite effect on different portions of the same gene. We characterized the mosquito E75, a key early gene mediating 20E action. Expression of three E75 isoforms correlates with activation and maintenance of vitellogenic genes. We plan to elucidate the molecular mechanisms through which the long-term sustained expression of E75 in conjunction with the stimulation of vitellogenic genes is achieved. The fat body and ovary become competent to respond to 20E activation as a result of post-eclosion development. A nuclear receptor FTZ-F1 plays an important role in competence acquisition. We will investigate the role of this factor in 20E activation of early and target genes, and the role of juvenile hormone in the acquisition of vitellogenic 20E response competence. The requirement of blood feeding for initiation of vitellogenesis is assured by the blockage of the regulatory hierarchy controlling vitellogenic gene activation. At the state-of arrest, Ultraspiracle (USP) is bound to a repressor, HR38. We will search for a specific signal that maintains this heterodimeric association. We have also identified the Adrenodoxin Reductase gene, encoding a key steroidogenic enzyme expressed in vitellogenic ovary, and will begin to dissect the mechanisms through which its activity is controlled by ecdysiotropic neuropeptide stimulation. Accomplishment of this proposed research will result in a deeper understanding of the action of ecdysteroids in the regulation of egg maturation in mosquitoes and may lead to the development of novel approaches to mosquito control.